Both androgens and estrogens contribute to the maintenance of bone mass during adult life in men, but the cell targets and molecular mechanisms of these effects remain poorly understood. During the preceding funding period, we found that the effects of androgens on cancellous bone result from AR signaling in cells of the mesenchymal lineage leading to a decrease in osteoclasts; and orchidectomy (ORX) increases soluble RANKL levels as well as the number of B-lymphocytes in the murine bone marrow. RANKL derived from osteocytes is critical for cancellous bone remodeling and B-lymphocyte-derived RANKL contributes to the loss of cancellous bone mass in ovariectomized (OVX) mice. In cortical bone, however, the effects of androgens do not require AR signaling in any cell type of the mesenchymal lineage, nor do they require ER? signaling downstream of committed osteoblast progenitors targeted by Osx1-Cre, or AR or ER? signaling in the osteoclast lineage. Estrogens, nonetheless, attenuate endocortical resorption in females by ER? signaling in uncommitted mesenchymal progenitors targeted by Prx1-Cre. In addition, the mRNA and secreted protein levels of SDF1 ? a chemotactic cytokine of the CXC family that is abundantly expressed in Prx1-Cre targeted cells and promotes osteoclast generation ? are higher in GFP-tagged ER? null cells as well as bone marrow cell cultures from female ER?f/f;Prx1-Cre mice as compared to identical cultures from littermate controls; and so is osteoclastogenesis in co-cultures of ER? null BM stromal or calvaria cells with macrophages. Furthermore, both OVX and ORX increase SDF1 in the bone marrow plasma, administration of E2 to ORX mice prevents this increase, and E2, but not DHT, prevents cortical bone loss in ORX mice. Finally, we found that the OVX- or ORX-induced cortical bone loss is prevented by restraining H2O2 generation in osteoclast mitochondria; and the loss of cortical bone mass in OVX mice is prevented by a 17?-E2 dendrimer conjugate (EDC), incapable of stimulating nuclear- initiated actions of ER?. Based on these advances, we will test the interrelated hypotheses that: in males, the protective effects of androgens on cancellous bone result from AR signaling in osteocytes, B-lymphocytes, or both. These actions result in the attenuation of RANKL and thereby attenuation of the number of cancellous osteoclasts. The protective effects of androgens against endocortical resorption result, at least in part, from ER?- mediated actions (upon aromatization of androgens to estrogens) on Prx1-Cre targeted uncommitted mesenchymal progenitors. These actions repress SDF1 production, thereby attenuating osteoclast formation and homing at the endosteal surfaces. The suppressive effect of estrogens on SDF1 production ultimately leads to the restraining of H2O2 accumulation in osteoclasts and results from non-nuclear-initiated signaling of the ER?. To advance these hypotheses we will try to elucidate the mechanism of the protective effects of androgens on cancellous bone by determining the role of osteocyte- and B-lymphocyte-derived RANKL as well as the role of the B lymphocyte AR in these effects. To do this we will study: i) mice in which RANKL is deleted from mature osteoblasts/osteocytes (RANKLf/f;Dmp1-Cre), ii) mice in which both AR and RANKL are deleted from osteoblasts/osteocytes (RANKLf/f;ARf/y;Dmp1-Cre), iii) mice in which RANKL is deleted from B-lymphocytes (RANKLf/f;CD19-Cre), and iv) mice in which AR is deleted from B-lymphocytes (ARf/y;CD19-Cre). In addition, we will elucidate the mechanism of the protective effects of androgens on cortical bone by investigating: a) whether down-regulation of SDF1 in uncommited mesenchymal progenitors is responsible for some of these effects using: i) mice in which SDF1 is deleted in Prx1-Cre targeted cells, and ii) mice in which the SDF1 receptor, CXCR4, is deleted in LysM-Cre targeted cells; and b) whether the effect of androgens results from ER?-mediated actions upon androgen aromatization to estrogens using ER?f/f;Prx1-Cre mice. Using co-cultures of stromal and hematopoietic cells, we will also examine whether SDF1 promotes osteoclastogenesis via H2O2; and whether the effect of estrogens on SDF1 results from non-nuclear-initiated signaling of the ER?.